An essential step in neurotransmission is the regulated release of neurotransmitter from synaptic vesicles. To maintain signaling in response to repeated stimulation, synaptic vesicles must be recycled and reused. This process requires that, following fusion, the membrane and proteins composing a vesicle be sorted from plasma membrane components and retrieved by endocytosis. Several endocytic pathways that are mobilized under different levels of activity have been proposed, although the consequences of recycling via different pathways are presently unknown. These pathways operate at different rates, which may affect the ability of the synapse to respond to continued stimulation. In addition, it is possible that the mode of endocytosis may regulate the propensity for future release of the resulting vesicles by targeting vesicles to. different functional pools. Alternatively, since different adaptor proteins have been associated with different endocytic routes, the pathway utilized may regulate the protein composition, and thus the functional properties, of the resulting vesicles. This idea is consistent with the finding that many synaptic vesicle proteins contain internalization motifs that may allow independent sorting. One synaptic vesicle protein that contains such a motif is the vesicular glutamate transporter VGLUT1. VGLUT1 also contains two Cterminal polyproline domains that may regulate the mode of endocytosis used by the transporter. Studying these domains may yield crucial insight into the molecular mechanisms and functional consequences of recycling by these different endocytic pathways. Since these domains are not present in any other vesicular neurotransmitter transporters, this additional regulation of VGLUT1 may explain the unique functional properties of VGLUT1 synapses such as a higher capacity for synaptic plasticity and an increased resistance to synaptic depression. I will therefore study the role of the first polyproline domain (PP1) in VGLUT1 endocytosis (Aim 1), determine whether VGLUT1 recycles by a pathway dependent on the APS adaptor protein (Aim 2), and examine whether the Nck1/2 scaffolding protein mediates the role of PP1 (Aim 3). I will address these questions through a combination of molecular biology, biochemistry, electron microscopy, and live cell neuronal imaging. PUBLIC HEALTH RELEVANCE: Release of glutamate at the nerve terminal requires transport of glutamate into synaptic vesicles by VGLUT1-3. Glutamate signaling has been implicated in schizophrenia, and drugs targeting glutamate release have recently emerged as a potential new class of antipsychotic. In addition, due to the key role of glutamate signaling in the central nervous system, it has also been implicated in other conditions such as anxiety disorders, epilepsy, and neuronal degeneration.